Compounds as modulators of a mutant cftr protein and their use for treating diseases associated with cftr protein malfunction

ABSTRACT

An exemplary embodiment relates to novel protein modulators capable of altering function of the mutant CFTR protein and their use for treating diseases associated with CFTR protein malfunction. An exemplary embodiment provides compositions, pharmaceutical preparations and methods of correcting the cellular alteration of a mutant CFTR protein wherein the CFTR mutation is a mutation ΔF508-CFTR, or another mutation of class II.

TECHNICAL FIELD

The exemplary techniques disclosed herein relate to novel proteinmodulators capable of altering function of the mutant CFTR protein andtheir use for treating diseases associated with CFTR proteinmalfunction. Exemplary embodiments provide compositions, pharmaceuticalpreparations and methods of correcting the cellular alteration of amutant CFTR protein wherein the CFTR mutation is a mutation ΔF508-CFTR,or another mutation of class II.

BACKGROUND

Cystic fibrosis (also known as CF or mucoviscidosis) is one of the mostcommon, fatal genetic diseases in humans. CF is an inherited autosomalrecessive genetic disease that affects around 1 child in 2,500 livebirths. CF is caused by mutations in the cftr gene that encodes thecystic fibrosis transmembrane conductance regulator (CFTR protein) withactivity as an epithelial chloride ion channel. As a result of impairedfunction of this protein, severe symptoms associated with respiratoryand digestive systems and male reproductive system appear. To date, morethan 1600 mutations in CFTR gene have been identified and described.

The CFTR gene mutations were classified into five classes based on themolecular mechanisms leading to the CFTR protein malfunction. The classI mutations contribute to the formation of proteins with incompletelength and usually involve the complete loss of its activity (e.g.G542X). Mutation in the class II lead to abnormal maturation of proteinsin the endoplasmic reticulum and Golgi apparatus. The effect of thesemutations is premature degradation of the protein. Hence, CFTR does notreach the cell membrane where it should perform its function (eg, ΔF508,ΔI507, S549R). The gene product having mutations of class III isproperly synthesized, transported and incorporated into the cellmembrane, but has decreased activity caused by abnormal regulation ofthe protein.

These mutations are frequently situated within one of the nucleotidebinding domain. (eg. G551D/S). Mutations of class IV cause anomalies inthe structure of the transmembrane protein and thereby reduce theconduction of chloride channel (e.g. R117H, R334W). Mutations alteringthe stability of mRNA represent a class V of the mutations of the CFTRgene (3849+10 kbC->T,5T).

The most prevalent mutation present in at least one allele inapproximately 90% of patients is a deletion of phenylalanine at position508 of the CFTR amino acid sequence (ΔF508 CFTR). This is a classicexample of class II mutation that causes premature degradation of theprotein. This mutation is associated with water-electrolyte disturbances(among others with chloride anion flux out of a cell across the plasmamembrane and the movement of sodium ions into the cell) and results inthe appearance of pathological symptoms. Some of the most severesymptoms include congestion and increased mucus viscosity in the upperand lower airways leading to lung damage. These conditions create afavorable environment for development of bacterial infections caused bye.g. Pseudomonas aeruginosa. Moreover, malfunction of CFTR protein leadsto obstruction of exocrine pancreatic ducts and related digestivedisorders.

CFTR is a glycoprotein with 1480 amino acids and classified as an ABC(ATP-binding cassette) transporter. The protein consists of fivedomains. There are two nucleotide binding domains (NBD1 and NBD2),regulatory domain (RD) and two transmembrane domains (TMD1 and TMD2).The protein activity is regulated by cAMP-dependent Protein Kinase (PKA)which catalyze phosphorylation of regulatory domain (RD) and also bybinding of two ATP molecules to NBD1 and NBD2 domains.

The disclosure in the patent application WO2007075901 (publ. 2007 Jul.5) relates to prodrugs of modulators of ABC transporters, particularly,CFTR modulators, compositions thereof, and methods therewith. Anexemplary embodiment also relates to methods of treating ABC transportermediated diseases using such modulators.

In U.S. Patent Publication No. 20080319008, compounds that increaseactivity (ion transport) of a mutant CFTR protein, and uses thereof aredescribed. The disclosure also provides compositions, pharmaceuticalpreparations and methods increasing ion transport activity of a mutantCFTR protein, i.e. ΔF508 CFTR, G551D-CFTR, G1349D-CFTR or D1152H-CFTR,that are suitable in treating cystic fibrosis (CF). The compositions andpharmaceutical preparations of the disclosure may comprise one or morephenylglycine-containing compounds or sulfonamide-containing compoundsor an analog or derivatives thereof.

In a publication WO2009051910, compounds that increase ion transportactivity of a mutant CFTR protein, and uses thereof are described. Thedisclosure provides compositions, pharmaceutical preparations andmethods for increasing activity of a mutant-CFTR. The compositions,pharmaceutical preparations and methods are notable for the study andtreatment of disorders associated with mutant-CFTR, such as cysticfibrosis. The compositions and pharmaceutical preparations of thedisclosure may comprise one or more phenylglycine-containing compounds,or an analog or derivative thereof.

U.S. Pat. No. 5,948,814 describes the use of genistein compound fortreatment of CF. A method of treating cystic fibrosis by generating CFTRfunction in cells containing mutant CFTR and the therapeutic compositionfor treatment are described. The method of treatment comprisesadministering an effective amount of genistein, or genistein analoguesand derivatives, to a patient afflicted with cystic fibrosis.

In U.S. Patent Publication No. 20040006127, a method for activation ofthe chloride is described. Fluorescein and derivatives have use in thetreatment of a disease condition of a living animal body, includinghuman, which disease is responsive to the activation of the CFTRchloride channels, for instance cystic fibrosis, disseminatedbrocheiectasis, pulmonary infections, chronic pancreatitis, maleinfertility and long QT syndrome.

In U.S. Patent Application No. 20080318984, compounds for correction ofthe cellular alteration of a mutant CFTR protein and uses thereof aredescribed. The disclosure provides for compositions, pharmaceuticalpreparations and methods for correcting cellular processing of amutant-CFTR protein (e.g., ΔF508 CFTR) that are notable for thetreatment of cystic fibrosis. The compositions and pharmaceuticalpreparations of the disclosure may comprise one or moreaminobenzothiazole-containing compounds, aminoarylthiazole-containingcompounds, quinazolinylaminopyrimidinone-containing compounds,bisaminomethylbithiazole-containing compounds, orphenylaminoquinoline-containing compounds, or an analog or derivativethereof.

In a publication WO2009051909, compounds that improve the cellularalteration of a mutant CFTR protein and uses thereof are described. Thedisclosure provides compositions, pharmaceutical preparations andmethods for increasing activity of a mutant-CFTR. The compositions,pharmaceutical preparations and methods are notable for the study andtreatment of disorders associated with mutant-CFTR, such as cysticfibrosis. The compositions of the disclosure may comprise one or morebithiazole-containing compounds, or an analog or derivative thereof.

Phenylalanine 508 in CFTR protein occurs on the surface of NBD1 domainof CFTR. Current structural and biophysical studies reveal nosignificant differences between wild-domain protein, and ΔF508 mutantdomain that may affect the folding kinetics and thermodynamic stabilityof CFTR protein. Solved crystal structures of both domains show onlyslight differences in the reorganization of the amino acids located nearthe site, which should be occupied by F508.

Each of the forgoing patents and publications are incorporated herein byreference in their entirety.

SUMMARY

The object of exemplary embodiments is to provide compositions,pharmaceutical preparations and methods of correcting the cellularprocessing of mutant CFTR protein. F508 deletion has minimal effect onthe structure of NBD1 domain as observed in the results of X-rays, andcannot explain the dramatic difference in the behavior of mutant andnative forms of CFTR protein in the cell. For the purposes of anexemplary embodiment, the structural data of both forms of protein weresubjected to computer simulation designed to determine the dynamicproperties of NBD1. In an exemplary embodiment the molecular dynamicsmethods have been used. This method is based on an iterative calculationof the interactions between the atoms forming the simulated system andsolving equations of motion. These simulations (for both studied formsof NBD1) results in sets of structures that can be adopted by the targetprotein according to the initial physical assumptions—the so-calledtrajectories.

Based on the analysis of molecular dynamics trajectories of the twodomains it is possible to isolate a mutant protein conformation, whichdiffers significantly from the conformational states adopted by the wildprotein. The conformation possesses the two major pockets on the surfaceof the protein located on both sides of the ATP binding site. Thestructure of protein in this conformation was used to develop compoundsfor the correction of ΔF508-CFTR activity.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 indicated effects of different compounds on iodide efflux at 1 μMin ΔF508-CFTR HeLa cells.

FIG. 2 indicates iodide efflux in untreated WT-CFTR HeLa cells.

FIG. 3 indicates iodine efflux in ΔF508-CFTR HeLa cells with forskolinalone, forskolin plus genistein or forskolin plus other compounds.

FIG. 4 indicates the impact of correctors on ΔF508-CFTR maturation andcell localization.

FIG. 5 indicates synergistic effects of active compounds on iodideefflux.

FIG. 6 indicates current-voltage relationship for cAMP-dependentchloride currents in HeLa cells.

FIG. 7 indicates effects of different compounds on iodide efflux.

FIG. 8 indicates the effect of 73100 plus 118208 molecules on nasalpotential difference (6VTE) in 6F508/M508 mice.

DETAILED DESCRIPTION

Exemplary embodiments will be described. Various modifications,adaptations or variations of the exemplary embodiments described hereinmay become apparent to those skilled in the art as such are disclosed.It will be understood that all such modifications, adaptations orvariations that rely upon the teachings hereof, and through which theseteachings have advanced the art, are considered to be within the scopeand spirit of the disclosure presented herein.

The methods and compositions of the exemplary embodiments may suitablycomprise, consist of, or consist essentially of the components,ingredients, elements, steps and process delineations described herein.The embodiments illustratively disclosed herein suitably may bepracticed in the absence of any element, process step, or ingredientwhich is not specifically disclosed herein.

Unless otherwise stated, all percentages, parts, and ratios expressedherein are based upon weight of the total compositions.

The headings provided herein serve to illustrate, but not to limit theteachings herein in any way or manner.

An exemplary embodiment is a compound of general formula (I):

its tautomers, E and Z geometrical isomers, optically active forms suchas enantiomers, diastereomers and their racemate forms or a mixture ofstereoisomeric forms or its pharmaceutically acceptable salts thereof orcomplexes thereof;wherein Z¹ is independently selected from the group consisting of:—C_(n)H_((2n))—, which is branched or unbranched wherein n is an integerfrom 1 to 5; —C_(n)H_((2n−2))— in E or Z geometrical conformation whichis branched or unbranched wherein n is an integer from 2 to 5;—C_(n)H_((2n−4))— which is branched or unbranched wherein n is aninteger from 2 to 5; —CR′H—, —C₂H₃R′—, E or Z—C₂HR′—, —C₃H₅R′—, E orZ—C₃H₃R′—, —OCH₂—, —CH₂O—, —NR″CH₂—, —CH₂NR″—; wherein R′ isindependently selected from the group consisting of: —H, halogen, —NH₂,—OH, —CN, CF₃, —CHF₂, —CH₂F, —SH, —SCN, —CH₃, —C₂H₅; wherein R″ isindependently selected from the group consisting of: —H, —CH₃, —C₂H₅;wherein R¹ and R² are independently selected from the group consistingof aromatic ring or heteroaromatic ring,as a modulator of a mutant CFTR protein for use in the treatment ofcystic fibrosis.R¹ and R² are independently selected from the group of sub-formula (Ia):

-   -   wherein A₁, A₂, A₃, A₄, A₅, A₆ is independently selected N or C        atoms wherein ring contain 0-3 nitrogen atoms;    -   wherein E¹, E², E³, E⁴, E⁵ represents optional substituents,        which are selected from:    -   —OR_(B), —OC(═O)R_(C), —OC(═O)OR_(B), —OC(═O)N(R_(A))R_(A)′,        —C(═O)R_(C), —C(═O)N(R_(A))R_(A)′, —C(═O)N(OR_(B))R_(A),        —C(═O)OR_(B), —C(═S)R_(C), —C(═O)C(═O)R_(C), —CH₂OR_(B),        —CH₂CH₂OR_(B), —CH₂N(R_(A))R_(A)′, —CH₂CH₂N(R_(A))R_(A)′,        —CH₂OCH₂R_(C), —CH₂N(R_(A))CH₂R_(C), —SR_(D), —S(═O)R_(D),        —SO₂R_(D), —SO₂N(R_(A))R_(A)′, —SO₃R_(B), —N(R_(A))C(═O)R_(C),        —N(R_(A))C(═O)OR_(B), —N(R_(A))C(═O)N(R_(A)′)R_(A)″,        —N(R_(A))SO₂R_(D), —N(R_(A))SO₂N(R_(A)′)R_(A)″, —N(R_(A))R_(A)′,        —N(R_(A))C(═O)R_(C), —N(R_(A))C(═O)OR_(B),        —N(R_(A))N(R_(A)′)R_(A)″, —N(R_(A)′)N(R_(A))C(═O)R_(C), —NO₂,        —CN, —CF₃, —CHF₂, —CH₂F, —NH₂, —SCN, —SO₂CN, —F, Cl, —Br, —I,        —PO₃H₂, —OPO₃H₂, —C_(n)H_(2n)R_(C) which is branched or        unbranched wherein n is an integer from 1 to 5;        —C_(n)H_((2n−2))R_(C) in E or Z geometrical conformation which        branched or unbranched wherein n is an integer from 2 to 5;        —C_(n)H_((2n−4))R_(C) which is branched or unbranched wherein n        is an integer from 2 to 5;    -   wherein R_(A), R_(A)′, R_(A)″ are each independently selected        from the group consisting of: —H, lower alkyl group, —CN, —CF₃,        —CHF₂, —CH₂F, —OH;    -   wherein R_(B) is independently selected from the group        consisting of: —H, lower alkyl group, —CN, —CF₃, —CHF₂, —CH₂F,        —CH₂Cl, —CH₂Br, —CH₂I;    -   wherein R_(C) is independently selected from the group        consisting of: —H, lower alkyl group, —CN, —CF₃, —CHF₂, —CH₂F,        —CH₂Cl, —CH₂Br, —CH₂I, —F, —Cl, —Br, —I, —NH₂,    -   wherein R_(D) is independently selected from the group        consisting of: —H, lower alkyl group;

An exemplary embodiment of the compound is represented by the followingstructures:

An exemplary embodiment of the compound has effect on mutant CFTRprotein, wherein said CFTR mutation is a mutation ΔF508-CFTR, or anothermutation of class II and where a mutation ΔF508-CFTR, or anothermutation of class II are involved in CFTR protein malfunction.

In an exemplary embodiment the CFTR protein malfunction occurs in theprotein associated with the disease cystic fibrosis.

A further exemplary embodiment is a modulator according to the above,for use in the treatment of cystic fibrosis wherein it has effect onCFTR-dependent ion transport across cellular membrane and/or it has theability to increase the number of mutant CFTR proteins that reach thecell surface.

An exemplary embodiment is used in the treatment of cystic fibrosiswherein it has stabilizing effect on the structure of the mutant CFTRprotein and/or blocks the interaction with cellular proteins responsiblefor the premature degradation of mutant CFTR

An exemplary embodiment is used in the treatment of cystic fibrosiswherein it has effect on mutant CFTR protein, wherein said CFTR mutationis a mutation ΔF508-CFTR, or another mutation of class II.

An exemplary embodiment are compounds, modulators of a mutant CFTRprotein, of general formula (II):

its tautomers, E and Z geometrical isomers, optically active forms suchas enantiomers, diastereomers and their racemate forms or a mixture ofstereoisomeric forms or its pharmaceutically acceptable salts thereof orcomplexes thereof;wherein Q¹ and Q² are independently selected from the group consistingof: C, CH, N, NH;wherein A is a fused five-membered ring having 0-3 independentlyselected heteroatoms wherein the heteroatoms comprise nitrogen, sulfuror oxygen;wherein R⁴, R⁵ and R⁶ represent optional substituents, which areindependently selected from: —OR_(B), —OC(═O)R_(C), —OC(═O)OR_(B),—OC(═O)N(R_(A))R_(A)′, —C(═O)R_(C), —C(═O)N(R_(A))R_(A)′,—C(═O)N(OR_(B))R_(A), —C(═O)OR_(B), —C(═S)R_(C), —C(═O)C(═O)R_(C),—CH₂OR_(B), —CH₂CH₂OR_(B), —CH₂N(R_(A))R_(A)′, —CH₂CH₂N(R_(A))R_(A)′,—CH₂OCH₂R_(C), —CH₂N(R_(A))CH₂R_(C), —SR_(D), —S(═O)R_(D), —SO₂R_(D),—SO₂N(R_(A))R_(A)′, —SO₃R_(B), —N(R_(A))C(═O)R_(C),—N(R_(A))C(═O)OR_(B), —N(R_(A))C(═O)N(R_(A)′)R_(A)″, —N(R_(A))SO₂R_(D),—N(R_(A))SO₂N(R_(A)′)R_(A)″, —N(R_(A))R_(A)′, —N(R_(A))C(═O)R_(C),—N(R_(A))C(═O)OR_(B), —N(R_(A))N(R_(A)′)R_(A)″,—N(R_(A)′)N(R_(A))C(═O)R_(C), —CN, —CF₃, —CHF₂, —CH₂F, —NH₂, —SCN,—SO₂CN, —F, Cl, —Br, —I, —PO₃H₂, —OPO₃H₂, which may be optionallypreceded by: —C_(n)H_((2n−1))R_(C) which is branched or unbranchedwherein n is an integer from 1 to 4; —C_(n)H_((2n−3))R_(C) in E or Zgeometrical conformation which is branched or unbranched wherein n is aninteger from 2 to 5; —C_(n)H_((2n−5))R_(C) which is branched orunbranched wherein n is an integer from 2 to 5;

-   -   wherein Z² is selected from: a single bond, —N(R_(A)′)—, —S—,        —S-alkil-, —O—, —O-alikil-, —C(═O)—, —S(═O)—, —OC(═O)—,        —C(═O)N(R_(A)′)—, —OC(═O)N(R_(A)′)—, —C(═O)O—, —SO₂—,        —SO₂N(R_(A)′)—, —N(R_(A)′)SO₂—, —N(R_(A)′)SO₂N(R_(A)″)—, —CH₂O—,        —N(R_(A)′)C(═O)—, —N(R_(A)′)C(═O)O—, —N(R_(A)′)C(═O)N(R_(A)″)—,        —C(═O)C(═O)—, —N(R_(A)′)C(═O)O—, —N(R_(A)′)N(R_(A))—,        —N(R_(A)′)N(R_(A)″)C(═O)—, —C(═O)N(R_(A)′)N(R_(A)″)—,        —CH₂N(R_(A)′)—, —CH₂CH₂O—, —CH₂CH₂N(R_(A)′)—, —CH₂OCH₂—,        —CH₂N(R_(A)′)CH₂—, —CH_(2n) which is branched or unbranched        wherein n is an integer from 1 to 5;        —C_(n)H_((2n−2)) in E or Z geometrical conformation which is        branched or unbranched wherein n is an integer from 2 to 5;        —C_(n)H_((2n−4)) which is branched or unbranched wherein n is an        integer from 2 to 5;        wherein R⁷ are independently selected from the group consisting        of: —H, aromatic ring or heteroaromatic ring;        wherein Z³ is selected from: a single bond, double bond,        —N(R_(A)′)—, —S—, —S-alkyl-, —O—, —O-alkyl-C(═O)—, —C(═S)—,        —OC(═O)—, —C(═O)N(R_(A)′)—, —OC(═O)N(R_(A)′)—, —C(═O)O—, —SO₂—,        —SO₂N(R_(A)′)—, —N(R_(A)′)SO₂—, —N(R_(A)′)SO₂N(R_(A)′)—, —CH₂O—,        —N(R_(A)′)C(═O)—, —N(R_(A)′)C(═O)O—, —N(R_(A)′)C(═O)N(R_(A)″)—,        —C(═O)C(═O)—, —N(R_(A)′)C(═O)O—, —N(R_(A)′)N(R_(A)″)—,        —N(R_(A)′)N(R_(A)″)C(═O)—, —C(═O)N(R_(A)′)N(R_(A)″)—,        —CH₂N(R_(A)′)—, —CH₂CH₂O—, —CH₂CH₂N(R_(A)′)—, —CH₂OCH₂—,        —CH₂N(R_(A)′)CH₂—, —C_(n)H_(2n)— which is branched or unbranched        wherein n is an integer from 1 to 5;        —C_(n)H_((2n−2)) in E or Z geometrical conformation which is        branched or unbranched wherein n is an integer from 2 to 5;        —C_(n)H_((2n−4)) which is branched or unbranched wherein n is an        integer from 2 to 5;        wherein R⁸ is selected from: H, O, S, aromatic ring or        heteroaromatic ring; —C_(B)H_((2n+1)) which is branched or        unbranched wherein n is an integer from 1 to 5; —C_(n)H_((2n−1))        in E or Z geometrical conformation which is branched or        unbranched wherein n is an integer from 2 to 5; —C_(n)H_((2n−3))        which is branched or unbranched wherein n is an integer from 2        to 5;        wherein R_(A), R_(A)′, R_(A)″ are each independently selected        from the group consisting of: —H, lower alkyl group, —CN, —CF₃,        —CHF₂, —CH₂F, —OH;        wherein R_(B) is independently selected from the group        consisting of: —H, lower alkyl group, —CN, —CF₃, —CHF₂, —CH₂F,        —CH₂Cl, —CH₂Br, —CH₂I;        wherein R_(e) is independently selected from the group        consisting of: —H, lower alkyl group, —CN, —CF₃, —CHF₂, —CH₂F,        —CH₂Cl, —CH₂Br, —CH₂I, —F, —Cl, —Br, —I, —NH₂;        wherein R_(D) is independently selected from the group        consisting of: —H, lower alkyl group;        wherein the 5-membered ring A is moiety selected from the group        consisting of:

and wherein compound6-[(4-oxo-1H-quinolin-3-yl)carbonylamino]-1H-indole-4-carboxylic acidethyl ester is excluded,as a modulator of a mutant CFTR protein, for use in the manufacture of amedicament for the treatment of diseases associated with CFTR proteinmalfunction.

An exemplary embodiment of the compound described above has the generalformula (IIa) or (IIb):

wherein Q¹, Q², Q³, Q⁴, Q⁵ represent optional substituents which areindependently selected from the group consisting of: —OR_(B),—OC(═O)R_(C), —OC(═O)OR_(B), —OC(═O)N(R_(A))R_(A)′, —C(═O)R_(C),—C(═O)N(R_(A))R_(A)′, —C(═O)N(OR_(B))R_(A), —C(═O)OR_(B), —C(═S)R_(C),—C(═O)C(═O)R_(C), —CH₂OR_(B), —CH₂CH₂OR_(B), —CH₂N(R_(A))R_(A)′,—CH₂CH₂N(R_(A))R_(A)′, —CH₂OCH₂R_(C), —CH₂N(R_(A))CH₂R_(C), —SR_(D),—S(═O)R_(D), —SO₂R_(D), —SO₂N(R_(A))R_(A)′, —SO₃R_(B),—N(R_(A))C(═O)R_(C), —N(R_(A))C(═O)OR_(B),—N(R_(A))C(═O)N(R_(A)′)R_(A)″, —N(R_(A))SO₂R_(D),—N(R_(A))SO₂N(R_(A)′)R_(A)″, —N(R_(A))R_(A)′, —N(R_(A))C(═O)R_(C),—N(R_(A))C(═O)OR_(B), —N(R_(A))N(R_(A)′)R_(A)″,—N(R_(A)′)N(R_(A))C(═O)R_(C), —NO₂, —CN, —CF₃, —CHF₂, —CH₂F, —NH₂, —SCN,—SO₂CN, —F, Cl, —Br, —I, —C_(n)H_(2n)R_(C) which is branched orunbranched wherein n is an integer from 1 to 5; —C_(n)H_((2n−2))R_(C) inE or Z geometrical conformation which is branched or unbranched whereinn is an integer from 2 to 5; —C_(n)H_((2n−4))R_(C) which is branched orunbranched wherein n is an integer from 2 to 5; PO₃H₂, —OPO₃H₂.

An exemplary embodiment of the compound may be represented by thefollowing structure:

An exemplary embodiment of the compound has effect on mutant CFTRprotein, wherein said CFTR mutation is a mutation ΔF508-CFTR, or anothermutation of class II and where a mutation ΔF508-CFTR, or anothermutation of class II. are involved in CFTR protein malfunction.

In an exemplary embodiment the CFTR protein malfunction occurs in theprotein associated with the disease cystic fibrosis.

A further exemplary embodiment is a modulator according to the above,for use in the treatment of cystic fibrosis wherein it has effect onCFTR-dependent ion transport across cellular membrane and/or it has theability to increase the number of mutant CFTR proteins that reach thecell surface.

An exemplary embodiment is used in the treatment of cystic fibrosiswherein it has stabilizing effect on the structure of the mutant CFTRprotein and/or blocks the interaction with cellular proteins responsiblefor the premature degradation of mutant CFTR

An exemplary embodiment is used in the treatment of cystic fibrosiswherein it has effect on mutant CFTR protein, wherein said CFTR mutationis a mutation ΔF508-CFTR, or another mutation of class II.

An exemplary embodiment is a compound of general formula (III):

its tautomers, E and Z geometrical isomers, optically active forms suchas enantiomers, diastereomers and their racemate forms or a mixture ofstereoisomeric forms or its pharmaceutically acceptable salts thereof orcomplexes thereof;wherein Z¹, Z², Z³, Z⁴, Z⁵, Z⁶, Z⁷ represents optional substituents,which are selected from substituents consisting at least one atomselected from the group consisting of: C, N, S, O, H, P, F, Cl, Br, I;wherein R⁴ represents optionally substituted moiety of formula (IIIa):

wherein R⁵ and R⁶ are optional substituents which are independentlyselected from the group consisting of: OH, NH₂, COOH, Cl, Br, I, CH₃,C₂H₅;and having a general formula (IIIb):

wherein R⁷ is an optional substituent which is independently selectedfrom the group consisting of: —F, —Cl, —Br, —I, —CH₃, —C₂H₅;wherein R⁸ is an optional substituent which is independently selectedfrom the group consisting of: —NH₂, —NHAr, —OH, —CH₂Ar, —C(═O)Ar, —OAr;wherein Ar is an aromatic group or heteroaromatic group;wherein Z¹, Z², Z³, Z⁴, Z⁵, Z⁶, Z⁷ represent optional substituents whichare independently selected from the group consisting of: —OR_(B),—OC(═O)R_(C), —OC(═O)OR_(B), —OC(═O)N(R_(A))R_(A)′, —C(═O)R_(C),—C(═O)N(R_(A))R_(A)′, —C(═O)N(OR_(B))R_(A), —C(═O)OR_(B), —C(═S)R_(C),—C(═O)C(═O)R_(C), —CH₂OR_(B), —CH₂CH₂OR_(B), —CH₂N(R_(A))R_(A)′,—CH₂CH₂N(R_(A))R_(A)′, —CH₂OCH₂R_(C), —CH₂N(R_(A))CH₂R_(C), —SR_(D),—S(═O)R_(D), —SO₂R_(D), —SO₂N(R_(A))R_(A)′, —SO₃R_(B),—N(R_(A))C(═O)R_(C), —N(R_(A))C(═O)OR_(B),—N(R_(A))C(═O)N(R_(A)′)R_(A)″, —N(R_(A))SO₂R_(D),—N(R_(A))SO₂N(R_(A)′)R_(A)″, —N(R_(A))R_(A)′, —N(R_(A))C(═O)R_(C),—N(R_(A))C(═O)OR_(B), —N(R_(A))N(R_(A)′)R_(A)″,—N(R_(A)′)N(R_(A))C(═O)R_(C), —NO₂, —CN, —CF₃, —CHF₂, —CH₂F, —NH₂, —SCN,—SO₂CN, —F, Cl, —Br, —I, —C_(n)H_(2n)R_(C) which is branched orunbranched wherein n is an integer from 1 to 5; —C_(n)H_((2n−2))R_(C) inE or Z geometrical conformation which is branched or unbranched whereinn is an integer from 2 to 5; —C_(n)H_((2n−4))R_(C) which is branched orunbranched wherein n is an integer from 2 to 5, —PO₃H₂, —OPO₃H₂;wherein R_(A), R_(A)′, R_(A)″ are each independently selected from thegroup consisting of: —H, lower alkyl group, —CN, —CF₃, —CHF₂, —CH₂F,—OH;wherein R_(B) is independently selected from the group consisting of:—H, lower alkyl group, —CN, —CF₃, —CHF₂, —CH₂F, —CH₂Cl, —CH₂Br, —CH₂I;wherein R_(C) is independently selected from the group consisting of:—H, lower alkyl group, —CN, —CF₃, —CHF₂, —CH₂F, —CH₂Cl, —CH₂Br, —CH₂I,—F, —Cl, —Br, —I, —NH₂;wherein R_(D) is independently selected from the group consisting of:—H, lower alkyl group,as a modulator of a mutant CFTR protein, for use in the manufacture of amedicament for the treatment of diseases associated with CFTR proteinmalfunction.

An exemplary embodiment of the compound is represented by the followingstructure:

An exemplary embodiment of the compound has effect on mutant CFTRprotein, wherein said CFTR mutation is a mutation ΔF508-CFTR, or anothermutation of class II and where a mutation ΔF508-CFTR, or anothermutation of class II are involved in CFTR protein malfunction.

In an exemplary embodiment the CFTR protein malfunction occurs in theprotein associated with the disease cystic fibrosis.

A further exemplary embodiment is a modulator according to the above,for use in the treatment of cystic fibrosis wherein it has effect onCFTR-dependent ion transport across cellular membrane and/or it has theability to increase the number of mutant CFTR proteins that reach thecell surface.

An exemplary embodiment is used in the treatment of cystic fibrosiswherein it has stabilizing effect on the structure of the mutant CFTRprotein and/or blocks the interaction with cellular proteins responsiblefor the premature degradation of mutant CFTR

An exemplary embodiment is used in the treatment of cystic fibrosiswherein it has effect on mutant CFTR protein, wherein said CFTR mutationis a mutation ΔF508-CFTR, or another mutation of class II.

An exemplary embodiment is a compound of general formula (IV):

its esters, ethers, tautomers, E and Z geometrical isomers, opticallyactive forms such as enantiomers, diastereomers and their racemate formsor a mixture of stereoisomeric forms or its pharmaceutically acceptablesalts thereof or complexes thereof;wherein E¹, E² represent substituents which are independently selectedfrom: H, —CH₃, —C₂; wherein E represents optional substituent selectedfrom: —CI, —F, —Br, —I, —CF₃, —CHF₂, —CH₂F, —CH₂Cl, —CH₂Br, —CH₂I,optionally substituted lower alkyl group;

An exemplary embodiment of the compound is represented by the followingstructure:

An exemplary embodiment of the compound has effect on mutant CFTRprotein, wherein said CFTR mutation is a mutation ΔF508-CFTR, or anothermutation of class II and where a mutation ΔF508-CFTR, or anothermutation of class II are involved in CFTR protein malfunction.

In an exemplary embodiment the CFTR protein malfunction occurs in theprotein associated with the disease cystic fibrosis.

A further exemplary embodiment is a modulator according to the above,for use in the treatment of cystic fibrosis wherein it has effect onCFTR-dependent ion transport across cellular membrane and/or it has theability to increase the number of mutant CFTR proteins that reach thecell surface.

An exemplary embodiment is used in the treatment of cystic fibrosiswherein it has stabilizing effect on the structure of the mutant CFTRprotein and/or blocks the interaction with cellular proteins responsiblefor the premature degradation of mutant CFTR

An exemplary embodiment is used in the treatment of cystic fibrosiswherein it has effect on mutant CFTR protein, wherein said CFTR mutationis a mutation ΔF508-CFTR, or another mutation of class II.

FIG. 1

The effects of different compounds on iodide efflux at 1 μM inΔF508-CFTR HeLa cells.

(a) bar graph showing the peak amplitudes of Fsk/Gsk dependent iodideeffluxes in cells treated by the different drugs as in A. Values aremean of 3 independent experiments. *p<0.05, **p<0.01.

(b) chemical structures of active correctors identified in silico

(c) examples of iodide efflux curves obtained in HeLa cells stablytransfected with ΔF508-CFTR and treated for 24 hours with 10 μM withdifferent compounds. CFTR dependent response was induced by 10 μMForskolin (Fsk)+30 μM Genistein (Gsk) as indicated by the horizontal barabove the traces.

(d) EC50 was determined for active compounds of pocket 2: 407882 and73100 and one of pocket 1: 130813, for 118208 EC50 could not beprecisely determined since the maximum of iodide efflux was not reachedeven at 100 μM (also shown).

FIG. 2

To test whether the compounds exhibit potentiator activity independentof their effect on CFTR trafficking, we examined iodide efflux inuntreated WT-CFTR HeLa cells. Compounds were added along with forskolinand their effects were compared to that of forskolin alone or forskolinplus genistein. Unlike genistein, all tested molecules induced an I⁻efflux greater than that of forskolin alone.

FIG. 3

Potentiation was also tested in ΔF508-CFTR HeLa cells treated for 2hours with miglustat to rescue ΔF508-CFTR. I⁻ efflux was stimulatedeither with forskolin alone, with forskolin plus genistein or forskolinplus the different compounds. As shown in the Figure, only genistein wasable to increase efflux, demonstrating the absence of potentiationactivity by The drugs.

FIG. 4

Impact of identified correctors on ΔF508-CFTR maturation and celllocalization.

(a) Effects of different compounds on CFTR processing. Representativeimmunoblots of WT-CFTR and ΔF508-CFTR proteins of the proteins from HeLacells treated with 1 μM of the different compounds for 24 hours with Mab24-1. The positions of the mature (band C) and immature (band B) formsof CFTR are indicated.

(b) Comparison of relative intensity (C/B+C) for WT-CFTR, ΔF508-CFTRalone and ΔF508-CFTR after correction with our molecules.

(c) Effects of the different compounds used at 1 μM on CFTRlocalization. Confocal imaging showing the plasma membrane localisationof WT-CFTR and intracellular localisation of ΔF508-CFTR. The effect ofdrugs is illustrated in panels c to f. Bars: 20 μM. Arrows indicatestaining of CFTR at the plasma membrane.

FIG. 5

Synergistic effect of active compounds on iodide efflux tested at 1 μM.

-   -   (a) Iodide efflux in response to 1 μM Forskolin (Fsk)+30 μM        Genistein (Gsk) as indicated by the horizontal bar above the        traces, for cells treated for 24 h with a tested compounds alone        and in combination as fallows (a) 407882, 118208, (b) 118208,        73100 (c) 407882, 37173. (d) bar graph showing the peak        amplitudes of Fsk/Gst dependent iodide effluxes in cells treated        by the different drugs as in. Values are mean of 3 independent        experiments. *p<0.05, **p<0.01

FIG. 6

(a)/(b) Current-voltage relationship for cAMP-dependent chloridecurrents in HeLa cells treated with 407882(12) plus 118208(6) compoundsat 1 μM.

FIG. 7

The effects of different compounds on iodide efflux at 1 μM in anepithelial serous cell line derived from a ΔF508 CF patient (CF-4KM)cells. The concentration-dependence has been shown for the most potentmolecule 407882

FIG. 8

The effect of 73100 plus 118208 molecules on nasal potential difference(ΔV_(TE)) in ΔF508/ΔF508 mice. Basal V_(TE) values and ΔV_(TE) changesinduced by perfusion of nasal epithelium with 100 μM amiloride,ΔV_(TEamil) were similar in mice treated with the two molecules or withliposomes alone. Perfusion of low Cl⁻ solution in 3 out of 5 micehyperpolarized V_(TE) by more than 2 mV (ΔV_(TEamil-lowCl)) i.e. thethreshold value established by us as significant effect of treatment.The CFTR-related current unmasked by CFTR inhibitor I_(lnh172)represents about 30% of (ΔV_(TEamil-lowCl)) (data not shown).

For a better understanding of some exemplary embodiments, the examplesof the subject matter are disclosed below.

Examples Materials and Methods Antibodies

The following antibodies were used: MAB25031 (clone 24-1, R&D systems,USA) and MM13-4 (Upstate,) monoclonal antibodies (mAb) for CFTRdetection; Fluorescent secondary antibodies Alexa 594 and 488 werepurchased from Molecular Probes (Cergy Pontoise, France)

Cell Culture

Stably transfected HeLa cells (with pTracer plasmid alone as a control(pTracer) or expressing WT-CFTR (spTCF-WT), ΔF508-CFTR s(pTCF-F508del)were provided by Pascale Fanen (Inserm U.468, Créteil, France) and grownas described in Bobadilla J L, Macek M, Jr., Fine J P, Farrell P M.Cystic fibrosis: a worldwide analysis of CFTR mutations—correlation withincidence data and application to screening. Hum Mutat, 2002 June;19(6):575-606. Briefly, HeLa cells were grown in Dulbecco's modifiedEagle's medium (DMEM) supplemented with 10% heat-inactivated FCS, 100U/ml penicillin, 100 μg/ml streptomycin and 250 jag/ml zeocin. Cultureswere done at 37° C. in a humidified incubator with 5% CO₂. Theexpression of WT-CFTR and ΔF508-CFTR in these cells was verified byimmunoprecipitation and immunocytochemistry throughout the study.Treatments with different molecules (at 1 and 10 μM) and vehicle weredone when cells reached 75% confluence.

CF-KM4 cell line, obtained by transformation of primary cultures of CFtracheal gland serous cells homozygous for the ΔF508 mutation by usingthe wild-type SV40 virus, were grown as described elsewhere in the art(Antigny, F. et al. Calcium homeostasis is abnormal in cystic fibrosisairway epithelial cells but is normalized after rescue of F508del-CFTR.Cell calcium 43, 175-83(2008)).

Immunoblot Experiments

Cells cultured in 75 cm² flasks were washed twice with ice cold PBS,scraped in 2 ml PBS and centrifuged at 600 g for 5 min. The pellets weresuspended in 300 μl RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 1%TritonX-100, 1% Na deoxycholate, 0.1% SDS, pH 7.5) at 4° C. for 30 minwith agitation After centrifugation at 15000 g for 30 min thesupernatants were processed for immunoblot experiments as previouslydescribed in the art (Baudouin-Legros, M. et al. Control of basal CFTRgene expression by bicarbonate-sensitive adenylyl cyclase in humanpulmonary cells. Cellular physiology and biochemistry: internationaljournal of experimental cellular physiology, biochemistry, andpharmacology 21, 75-86(2008)) with slight modifications.

The samples were resolved by 8% SDS-PAGE, transferred onto PVDFmembranes and analysis was performed following manufacturer'srecommendations for the Odyssey infrared imaging system (LI-CORBiosciences, NE, USA). Blot membranes were blocked with Odyssey buffer(ScienceTec, Paris, France) for 1 hour and hybridized using monoclonalanti CFTR Mab24-1 ( 1/1000). The proteins were visualized by incubationwith secondary antibodies ( 1/10000) and detected using ECL technique asdescribed in, Bensalem, N. et al. Down-regulation of theanti-inflammatory protein annexin A1 in cystic fibrosis knock-out miceand patients. Molecular & cellular proteomics: MCP 4, 1591-601(2005).

Immunofluorescence Staining

HeLa cells grown on glass coverslips were treated as above and asdescribed in Lipecka, J. et al., Distribution of ClC-2 chloride channelin rat and human epithelial tissues. American journal of physiology.Cell physiology 282, C805-16(2002). Cells were fixed with 4%formaldehyde and permeabilized with 0.1% Triton in PBS. Cells wereblocked with 1% bovine serum albumine in PBS/Triton and incubated at 4°C. overnight with the primary antibodies, 24-1 (1:300). After washingand blocking with 5% normal goat serum, cells were incubated with thesecondary antibodies. Glass coverslips were mounted using theVectashield mounting medium (Vector laboratories) and examined byconfocal laser microscopy (Zeiss, LSM 510).

Iodide Efflux Experiments

CFTR chloride channel activity was assayed by measuring iodide (¹²⁵I)efflux from transfected CHO cells as described previously in the art,Marivingt-Mounir, C, et al., Synthesis, SAR, crystal structure, andbiological evaluation of benzoquinoliziniums as activators of wild-typeand mutant cystic fibrosis transmembrane conductance regulator channels.J Med Chem, 2004. 47(4): p. 962-72. Marivingt-Mounir, C, et al.,Synthesis, SAR, crystal structure, and biological evaluation ofbenzoquinoliziniums as activators of wild-type and mutant cysticfibrosis transmembrane conductance regulator channels. J Med Chem, 2004.47(4): p. 962-72. Cells grown for 4 days in 96-well plates were washedtwice with 2 ml of modified Earle's salt solution containing 137 mMNaCl, 5.36 mM KCl, 0.4 mM Na₂HPO₄, 0.8 mM MgCl₂, 1.8 mM CaCl₂, 5.5 mMglucose, and 10 mM HEPES, pH 7.4. Cells were then incubated in the samemedium containing 1 mM KI (1 mCi of Na¹²⁵I/ml, NEN Life ScienceProducts) for 30 min at 37° C. After washing, cells were incubated with1 ml of modified Earle's salt solution. After 1 min, the medium wasremoved to be counted and was quickly replaced by 1 ml of the samemedium. This procedure was repeated every 1 min for 8 min. The firstthree aliquots were used to establish a stable baseline in efflux bufferalone. Medium containing cocktail aiming to increase intracellular cAMP(10 μM forskolin and 30 μM genistein) was used for next aliquots inorder to activate CFTR chloride channels. At the end of the incubation,the medium was recovered, and cells were solubilized in 1 N NaOH. Theradioactivity was determined using a g-counter (LKB). The total amountof ¹²⁵I (in cpm) at time 0 was calculated as the sum of cpm counted ineach 1-min sample plus the cpm in the NaOH fraction. The fraction ofinitial intracellular ¹²⁵I lost during each time point was determined,and time-dependent rates of ¹²⁵I efflux were calculated according to theart in, Becq, F., et al., Development of substitutedBenzo[c]quinolizinium compounds as novel activators of the cysticfibrosis chloride channel. J Biol Chem, 1999. 274(39): p. 27415-25, fromln(¹²⁵I_(t1)/¹²⁵I_(t2))/(t₁−t₂), where

¹²⁵It is the intracellular ¹²⁵I at time t; and

t₁ and t₂ are successive time points.

Curves were constructed by plotting rate of ¹²⁵I efflux versus time.Data are presented as the mean±S.E. of n separate experiments.

Differences were considered statistically significant using theStudent's t test when the p value was less than 0.05.

Whole Cell Patch-Clamp Recordings

Technique for patch-clamp recordings in the whole cell configuration hasbeen described, such as in Hinzpeter, A. et al. Association betweenHsp90 and the ClC-2 chloride channel upregulates channel function.American journal of physiology. Cell physiology 290, C45-56(2006) andTanguy, G. et al. CSN5 binds to misfolded CFTR and promotes itsdegradation. Biochimica et biophysica acta 1783, 1189-99(2008). Stablytransfected cells were plated in 35 mm cell culture plastic Petri dishesthat were mounted on the stage of an inverted microscope. Patch-clampexperiments were performed at room temperature with an Axopatch 200Aamplifier controlled by a computer via a digitdata 1440 interface (AxonIntruments, USA). Pipettes were pulled from hard glass (Kimax 51) usinga Setter micropipette puller and their tips were fire-polished. Currentrecordings were performed using the nystatin-perforated patch clampconfiguration. Nystatin stock solution (50 mg/ml) was prepared daily inDMSO. The stock solution was diluted (1:250) in the internal solutionwhich was then sonicated during 1 minute. The internal solutioncontained the following (in mM), 131 NaCl, 2 MgCl₂, and 10 Hepes-Na⁺, pH7.3, adjusted with NaOH. The bath solution contained (in mM): 150 NaCl,1 CaCl₂, 1 MgCl₂, 35 sucrose and 10 Hepes-Na⁺, pH 7.3, adjusted withNaOH.

Currents were recorded by application of regular voltage pulses of 60 mVamplitude during 1 second, from a holding potential of 0 mV, with aninterval of 3 seconds.

To establish I-V curves, regular voltage pulses were interrupted byseries of 9 voltage jumps (1-s duration each), toward membranepotentials between −100 and +80 mV. CFTR Cl− currents were activatedwith 200 m 8-(4-chlorophenylthio)-cAMP sodium salt (CPT-cAMP) plus 100μM 3-isobutyl-1-methylxanthine (IBMX).

When maximal stimulation was reached, cells were bathed with 5 μM of thespecific CFTRinhibitor, CFTR_(inh)-172, added to the CPT-cAMP solution.CFTR-currents were defined as the differences in current amplitudesrecorded during maximum stimulation by CPT-cAMP and after inhibition byCFTR_(inh)-172.

Nasal Potential Difference (NPD) Measurements

The method for nasal potential measurement was adapted and miniaturisedfrom the technique developed for young children as shown inSermet-Gaudelus, I. et al. Measurement of nasal potential difference inyoung children with an equivocal sweat test following newborn screeningfor cystic fibrosis. Thorax 65, 539-44(2010). Mice were anesthetized byan intraperitoneal injection of ketamine (133 mg/kg; IMALGENE 1000,MERIAL, France) and xylazine (13.3 mg/kg; Rompun 2%, BayerPharma,France). Mice were positioned on a 45° tilt board and a paper pad wasplaced under the nose to avoid mice quelling. A subcutaneous needle wasconnected to an Ag⁺/AgCl reference electrode by an agar bridge. Adouble-lumen polyethylene catheter (0.5 mm diameter) was inserted intoone nostril 4 mm depth. One lumen perfused by a Ringer solution (in mM:140 NaCl, 6 KCl, 10 Hepes, 10 Glucose, 1 MgCl2, 2 CaCl2, pH adjusted to7.4 with NaOH) at 0.15 mL/h is connected to a measuring Ag⁺/AgClelectrode. The two Ag⁺/AgCl electrodes were connected to ahigh-impedance voltmeter (LOGAN research Ltd, United Kingdom). Thesecond lumen perfused solution with the following sequence: (1) Ringersolution, (2) Ringer solution containing amiloride (inhibitor of Na⁺conductance, 100 μM), (3) Low Chloride Ringer solution, to unmask Cl⁻conductances (in mM: 140 Na gluconate, 6 K gluconate, 10 Hepes, 10Glucose, 1 MgCl₂, 6 Ca-gluconate, pH adjusted to 7.4 with NaOH), (4) LowChloride Ringer solution containing CFTR inhibitor-172 (5 μM,Calbiochem, Germany) to evaluate the participation of CFTR. Eachsolution was perfused at least 3 minutes, and 30 seconds stability wasrequired before perfusion switch. Steady state transepithelialpotential, V_(TE), ΔV_(TEAmil) (difference between V_(TE) andtransepithelial potential recorded after perfusion ofamiloride-containing solution), ΔV_(TEamilLowCl) (difference betweenV_(TE) and transepithelial potential recorded after perfusion with LowCl⁻ plus amiloride-containing solution) and ΔV_(TEamilLowCllnh-172)(difference between V_(TE) and after addition of CFTR inhibitor to theprevious solution) were the means of 30 values recorded duringstability.

MTT Cell Viability Assay

To determine cell viability the typical MTT assay was used. HeLa cellswere cultured in a 96-well plate and exposed to varying concentrationsof compounds disclosed herein for 24 h. After washing, MTT solution andmedium were then introduced. After incubation, the resultant formazancrystals were dissolved in dimethyl sulfoxide and the absorbanceintensity measured by a microplate reader at 570 nm.

Yellow MTT is reduced to purple formazan in living cells. The absorbanceof this colored solution can be quantified by measuring at a certainwavelength by a spectrophotometer. This conversion can be directlyrelated to the number of viable (living) cells.

Virtual Screening—Identification of Modulator Compounds

A database of a low molecular weight compounds was used in the virtualscreening process as a source of hits. Molecular docking program Dock6.1 was used to test a conformational space of small molecules insidetwo potential binding sites on the protein surface. Subsequently, allselected ligands and whole complexes were fully minimized in forcefield. At each step, a set of scoring functions was used for rating ofpotential

118208/NOP1.6/Pok 1c 2-[(3-nitrophenyl)methylsulfanyl]-3,7-dihydropurin-6-one

407882/NOP2.6/Pok 2f 2-[hydroxy(phenyl)phosphoryl]ethyl-phenylphosphinic acid

130813/NOP1.2/Pok1b 4-((6-chloro-2-methoxy-9-acridinyl)amino)-2-((4-methyl-1- piperazinyl)methyl)phenol

73100/Pok2e 2-(9H-fluoren-2- ylcarbamoyl)terephthalic acid

Results

Effect of drugs on iodide, I−, efflux

To test drug correction of ΔF508-CFTR trafficking and function weevaluated halide permeability by a macroscopic assay using a roboticcell-based methodology using the I⁻ efflux technique. In the firstseries of experiments, the potential corrector effects were tested by 24hour pre-treatment of ΔF508-CFTR HeLa cells with all compounds at 1 μMfollowed by measurements of cAMP-dependent radiolabel iodide efflux.Treatments with compounds 130813 and 118208 on pocket 1 and 73100 and407882 on pocket 2, lead to significant increase of cAMP-stimulatedradiolabel iodide efflux (FIG. 1a ), the most potent being 407882. Atthis low dose (1 μM) the increase in the cAMP-stimulated efflux waslower than that observed using 100 μM of the known corrector miglustat(27). Examples of I⁻ efflux stimulation after treatment with each of thefour active compounds are illustrated in FIG. 1b . cAMP-stimulated I⁻efflux was completely prevented when experiments were performed in thepresence of the CFTR channel blocker CFTR_(inh)-172.

We further tested the effect of the four compounds in a wide range ofconcentrations and determined EC₅₀ for pocket 1 compound 130813, and twopocket 2 compounds 407882 and 73100 at 1 μM, 10 μM and 844 nM,respectively (FIG. 1c ). The EC₅₀ for pocket 1-118208 could not beprecisely determined since the maximum iodide efflux was not reachedeven at 100 μM (FIG. 1d ). Notably, the effect of compound 407882 couldbe increased by 3-fold when used at 10 μM, reaching a stimulated effluxcomparable to the value observed for WT-CFTR (FIG. 2).

To test whether the compounds exhibit potentiator activity independentof their effect on CFTR trafficking, we examined iodide efflux inuntreated WT-CFTR HeLa cells. Compounds were added along with forskolinand their effects were compared to that of forskolin alone or forskolinplus genistein. Unlike genistein, all tested molecules induced an I⁻efflux greater than that of forskolin alone (FIG. 2). Potentiation wasalso tested in ΔF508-CFTR HeLa cells treated for 2 hours with miglustatto rescue ΔF508-CFTR. I⁻ efflux was stimulated either with forskolinalone, with forskolin plus genistein or forskolin plus the differentcompounds. As shown in FIG. 3, only genistein was able to increaseefflux, demonstrating the absence of potentiation activity by our drugs.

Effect of Drugs on CFTR Maturation

The efficacy of the four compounds as correctors for ΔF508-CFTRtrafficking was further validated by immunoblotting. We assumed thatdetection of a fully glycosylated band C suggests correct processing ofΔF508-CFTR. A representative immunoblot is shown in FIG. 4a . Anti-CFTRantibodies detect two bands in proteins derived from WT-CFTR cells,(line WT-CFTR in FIG. 4a ). The diffuse band of approximately 170 kDa(band C) corresponds to a mature, fully glycosylated protein that hasprocessed through the Golgi apparatus. The band below of about 145 kDacorresponds to the immature core-glycosylated protein located in theendoplasmic reticulum. In ΔF508-CFTR expressing cells, only the immatureprotein is detectable (line ΔF508 in FIG. 4a ). Band C was clearlydetectable in cells treated with 1 μM of compound 407882 as compared tountreated cells, whereas the signal at 170 kDa was not different fromDMSO treatment in cells treated with 1 μM of compounds 118208, or 130813or very slightly increased in cells treated by 1 μM of compound 73100.None of the compounds modified total protein expression. The relativeabundance of mature CFTR, expressed as the ratio of band C to bandC+band B is shown in FIG. 4b . Only compound 407882 increasedsignificantly the relative abundance of mature CFTR.

Effect of Drugs on CFTR Immunolocalization

FIG. 4c shows typical CFTR staining at the plasma membrane in WT-CFTRexpressing HeLa cells whereas ΔF508-CFTR was found throughout thecytoplasm. Treatment of cells for 24 hours with 1 μM of 407882 resultedin a clear CFTR staining at or near the plasma membrane, indicatingrescue of ΔF508-CFTR trafficking in agreement with immunoblotexperiments. When cells were treated by each of the three othercompounds, 118208, 73100 or 130813, a discrete punctuate staining at theplasma membrane was observed in a small fraction of cells, asillustrated for compound 118208 in FIG. 4 c.

Combined Effect of Compounds Binding to Different Pockets.

If two compounds are able to correct ΔF508-CFTR by binding to the sameprotein conformation but at different surface cavities their effectscould be additive or synergistic. We tested this hypothesis by twoindependent types of assays, namely iodide efflux and patch clamp. Theresults from iodide permeability tests (FIG. 5) showed that combinedtreatment of cells with compounds 118208 plus 407882 (FIG. 5a ) or with118208 plus 73100 (FIG. 5b ) at 1 μM of each, leads to greatercAMP-dependent anion fluxes than those observed with any of themolecules alone. In this series of experiments the compound 37173 (FIG.5c ) was used as a control as it did not induce any cAMP-stimulatediodide efflux at the same concentration. As shown in FIG. 5c ,co-treatment of ΔF508-CFTR HeLa cells with 37173 plus 407882 inducedcAMP-stimulated iodide efflux with an amplitude similar to 407882treatment alone. By contrast, co-treatment with compounds 118208 and407882 induced iodide efflux with an amplitude equal to the sum ofeffluxes induced by each compound, whereas a slight synergistic effectwas observed after treatment by 118208 plus 73100.

The activity of the different compounds was also evaluated inpatch-clamp experiments. FIG. 6a summarizes the mean values of currentamplitudes recorded at −60 mV in the different experimental conditions.CFTR-related current density (I_(ΔF508-CFTR); pA/pF) is defined ascAMP-stimulated current minus the current recorded after inhibition byCFTR inh-172 at 5 μM, and normalized to cell capacitance. I_(ΔF508-CFTR)was very low in untreated cells and stimulated by around 3-fold whencells were cultured at 27° C. for 24 hours before recordings. Treatmentof cells for 24 h with 1 μM of either 118208, 407882 or 73100 alone, didnot increase current amplitude as compared to their respective controls(data not shown). However, 24 h pre-treatment with 1 μM of 118208 plus407882 or 118208 plus 73100 showed a significant increase inI_(ΔF508-CFTR). Examples of linear I/V plots from cells pretreated by118208 plus 407882 before stimulation, in the presence of cptcAMP+IBMXand after inhibition by CFTRinh-172 are shown in FIG. 6 b.

Effects of 407882 and 118208 on CF-4KM Cells

The effects of the four molecules active in HeLa cells were next testedon CFTR-dependent iodide efflux in an epithelial serous cell linederived from a ΔF508 CF patient (CF-KM4) expressing low amounts ofendogenous ΔF508-CFTR. In these epithelial cells compounds 407882 and118208 were still able to induce significant cAMP-dependent iodideefflux (FIG. 7). However, it must be noted that 2 molecules correctingΔF508-CFTR in Hela cells (130813 and 73100) were not active in this cellline.

Effects of 73100 Plus 118208 on Nasal Potential Difference in ΔF508Mice.

Our results in cells suggested that the pairs of molecules acting ondifferent pockets display additive correcting effects. To test if thesemolecules are active in vivo, nasal potential difference (ΔV_(TE)) wasmonitored (as in Sermet-Gaudelus, I. et al., Measurement of nasalpotential difference in young children with an equivocal sweat testfollowing newborn screening for cystic fibrosis. Thorax 65,539-44(2010)) in ΔF508/ΔF508 mice treated intranasally for 24 hours with30 μl of 73100 plus 118208 molecules (0.1 μmol each) embedded inliposomes (5:1) or with liposomes alone. In ΔF508 mice, basal V_(TE)values and ΔV_(TE) changes induced by perfusion of nasal epithelium with100 μM amiloride, ΔV_(TEamil) were similar in mice treated with the twomolecules or with liposomes alone. By contrast, perfusion of low Cl⁻solution in 3 out of 5 mice hyperpolarized V_(TE) by more than 2 mV(ΔV_(TEamil-lowCl)) i.e. the threshold value established by us assignificant effect of treatment (manuscript in preparation). TheCFTR-related current unmasked by CFTR inhibitor I_(lnh172) representsabout 30% of (ΔV_(TEamil-lowCl)) (data not shown).

The following references are incorporated herein in their entirety:

-   1. Ollero M, Brouillard F, Edelman A. Cystic fibrosis enters the    proteomics scene: new answers to old questions. Proteomics, 2006    July; 6(14):4084-99.-   2. Riordan J R, Rommens J M, Kerem B, Alon N, Rozmahel R, Grzelczak    Z, et al. Identification of the cystic fibrosis gene: cloning and    characterization of complementary DNA. Science, 1989 Sep. 8;    245(4922):1066-73.-   3. Rommens J M, lannuzzi M C, Kerem B, Drumm M L, Melmer G, Dean M,    et al. Identification of the cystic fibrosis gene: chromosome    walking and jumping. Science, 1989 Sep. 8; 245(4922): 1059-65.-   4. Castellani C, Cuppens H, Macek M, Jr., Cassiman J J, Kerem E,    Durie P, et al. Consensus on the use and interpretation of cystic    fibrosis mutation analysis in clinical practice. J Cyst Fibros, 2008    May; 7(3): 179-96.-   5. Cystic Fibrosis Mutation Database    http://www.genet.sickkids.on.ca/cftr/app.-   6. Welsh M J, Smith A E. Molecular mechanisms of CFTR chloride    channel dysfunction in cystic fibrosis. Celi, 1993 Jul. 2; 73(7):    1251-4.-   7. Wilschanski M, Zielenski J, Markiewicz D, Tsui L C, Corey M,    Levison H, et al. Correlation of sweat chloride concentration with    classes of the cystic fibrosis transmembrane conductance regulator    gene mutations. J Pediatr, 1995 November; 127(5):705-10.-   8. Ward C L, Omura S, Kopito R R. Degradation of CFTR by the    ubiquitin-proteasome pathway. Cell, 1995 Oct. 6; 83(1):121-7.-   9. Bobadilla J L, Macek M, Jr., Fine J P, Farrell P M. Cystic    fibrosis: a worldwide analysis of CFTR mutations-correlation with    incidence data and application to screening. Hum Mutat, 2002 June;    19(6):575-606.-   10. Lewis H A, Zhao X, Wang C, Sauder J M, Rooney I, Noland B W, et    al. Impact of the deltΔF508 mutation in first nucleotide-binding    domain of human cystic fibrosis transmembrane conductance regulator    on domain folding and structure. J Biol Chem, 2005 January 14;    280(2): 1346-53.-   11. Schwiebert E M, Benos D J, Egan M E, Stutts M J, Guggino W B.    CFTR is a conductance regulator as well as a chloride channel.    Physiol Rev, 1999 January; 79(1 Suppl):S145-66.-   12. Reddy M M, Light M J, Cjuinton P M. Activation of the epithelial    Na+ channel (ENaC) requires CFTR Cl− channel function. Nature, 1999    Nov. 18; 402(6759):301-4.-   13. Ahmed N, Corey M, Forstner G, Zielenski J, Tsui L C, Ellis L, et    al. Molecular consequences of cystic fibrosis transmembrane    regulator (CFTR) gene mutations in the exocrine pancreas. Gut, 2003    August; 52(8): 1159-64.-   14. Riordan J R. Assembly of functional CFTR chloride channels. Annu    Rev Physiol, 2005; 67:701-18.-   15. Allen M P, Tildesley D J. Computer simulation of liquids:    Oxford, Clarendon Press; 1987.-   16. Jungas, T., et al., Glutathione levels and BAX activation during    apoptosis due to oxidative stress in cells expressing wild-type and    mutant cystic fibrosis transmembrane conductance regulator. J Biol    Chem, 2002. 277(31): p. 27912-8.-   17. Antigny, F. et al. Calcium homeostasis is abnormal in cystic    fibrosis airway epithelial cells but is normalized after rescue of    F508del-CFTR. Cell calcium 43, 175-83(2008).-   18. Baudouin-Legros, M. et al. Control of basal CFTR gene expression    by bicarbonate-sensitive adenylyl cyclase in human pulmonary cells.    Cellular physiology and biochemistry: international journal of    experimental cellular physiology, biochemistry, and pharmacology 21,    75-86(2008).-   19. Bensalem, N. et al. Down-regulation of the anti-inflammatory    protein annexin A1 in cystic fibrosis knock-out mice and patients.    Molecular & cellular proteomics: MCP 4, 1591-601(2005).-   20. Lipecka, J. et al. Distribution of ClC-2 chloride channel in rat    and human epithelial tissues. American journal of physiology. Cell    physiology 282, C805-16(2002).-   21. Marivingt-Mounir, C, et al., Synthesis, SAR, crystal structure,    and biological evaluation of benzoquinoliziniums as activators of    wild-type and mutant cystic fibrosis transmembrane conductance    regulator channels. J Med Chem, 2004. 47(4): p. 962-72.-   22. Becq, F., et al., Development of substituted    Benzo[c]quinolizinium compounds as novel activators of the cystic    fibrosis chloride channel. J Biol Chem, 1999. 274(39): p. 27415-25.-   23. Hinzpeter, A. et al. Association between Hsp90 and the ClC-2    chloride channel upregulates channel function. American journal of    physiology. Cell physiology 290, C45-56(2006).-   24. Tanguy, G. et al. CSN5 binds to misfolded CFTR and promotes its    degradation. Biochimica et biophysica acta 1783, 1189-99(2008).-   25. Sermet-Gaudelus, I. et al. Measurement of nasal potential    difference in young children with an equivocal sweat test following    newborn screening for cystic fibrosis. Thorax 65, 539-44(2010).-   26. Norez, C. et al. Maintaining low Ca2+ level in the endoplasmic    reticulum restores abnormal endogenous F508del-CFTR trafficking in    airway epithelial cells. Traffic (Copenhagen, Denmark) 7,    562-73(2006).-   27. Norez, C. et al. Rescue of functional delF508-CFTR channels in    cystic fibrosis epithelial cells by the alpha-glucosidase inhibitor    miglustat. FEBS letters 580, 2081-6(2006).

Of course these methods are exemplary and alterations thereto arepossible by those having skill in the relevant technology.

Thus the example embodiments and arrangements achieve improvedcapabilities, eliminate difficulties encountered in the use of priormethods and systems, and attain the desirable results described herein.

In the foregoing description, certain terms have been used for brevity,clarity and understanding. However, no unnecessary limitations are to beimplied therefrom because such terms are used for descriptive purposesand are intended to be broadly construed.

Moreover the descriptions and illustrations herein are by way ofexamples and the inventive scope is not limited to the features shownand described.

Further, it should be understood that features and/or relationshipsassociated with one embodiment can be combined with features and/orrelationships from other embodiments. That is, various features and/orrelationships from various embodiments can be combined in furtherembodiments. The inventive scope of the disclosure is not limited toonly the embodiments shown or described herein.

Having described the features, discoveries and principles of theexemplary embodiments, the manner in which they are utilized and carriedout, and the advantages and useful results attained, the new and usefularrangements, combinations, methodologies, structures, devices,elements, combinations, operations, processes and relationships are setforth in the appended claims.

We claim:
 1. A composition comprising: a modulator of a mutant CFTRprotein adapted for use in the manufacture of a medication for thetreatment of diseases associated with CFTR protein malfunctionincluding: a compound of general formula (I)

its tautomers, E and Z geometrical isomers, optically active forms suchas enantiomers, diastereomers and their racemate forms or a mixture ofstereoisomeric forms or its pharmaceutically acceptable salts thereof orcomplexes thereof; wherein Z¹ is independently selected from the groupconsisting of: —C_(n)H_((2n))—, which is branched or unbranched whereinn is an integer from 1 to 5; —C_(n)H_((2n−2))— in E or Z geometricalconformation which is branched or unbranched wherein n is an integerfrom 2 to 5; —C_(n)H_((2n−4))— which is branched or unbranched wherein nis an integer from 2 to 5; —CR′H—, —C₂H₃R′—, E or Z—C₂HR′—, —C₃H₅R′—, Eor Z—C₃H₃R′—, —OCH₂—, —CH₂O—, —NR″CH₂—, —CH₂NR″—; wherein R′ isindependently selected from the group consisting of: —H, halogen, —NH₂,—OH, —CN, CF₃, —CHF₂, —CH₂F, —SH, —SCN, —CH₃, —C₂H₅; wherein R″ isindependently selected from the group consisting of: —H, —CH₃, —C₂H₅;wherein R¹ and R² are independently selected from the group consistingof aromatic ring or heteroaromatic ring.
 2. The composition of claim 1,wherein R¹ and R² are independently selected from the group ofsub-formula (Ia):

wherein A₁, A₂, A₃, A₄, A₅, A₆ is independently selected N or C atomswherein ring contain 0-3 nitrogen atoms; wherein E, E², E³, E⁴, E⁵represents optional substituents, which are selected from: —OR_(B),—OC(═O)R_(C), —OC(═O)OR_(B), —OC(═O)N(R_(A))R_(A)′, —C(═O)R_(C),—C(═O)N(R_(A))R_(A)′, —C(═O)N(OR_(B))R_(A), —C(═O)OR_(B), —C(═S)R_(C),—C(═O)C(═O)R_(C), —CH₂OR_(B), —CH₂CH₂OR_(B), —CH₂N(R_(A))R_(A)′,—CH₂CH₂N(R_(A))R_(A)′, —CH₂OCH₂R_(C), —CH₂N(R_(A))CH₂R_(C), —SR_(D),—S(═O)R_(D), —SO₂R_(D), —SO₂N(R_(A))R_(A)′, —SO₃R_(B),—N(R_(A))C(═O)R_(C), —N(R_(A))C(═O)OR_(B),—N(R_(A))C(═O)N(R_(A)′)R_(A)″, —N(R_(A))SO₂R_(D),—N(R_(A))SO₂N(R_(A)′)R_(A)″, —N(R_(A))R_(A)′, —N(R_(A))C(═O)R_(C),—N(R_(A))C(═O)OR_(B), —N(R_(A))N(R_(A)′)R_(A)″,—N(R_(A)′)N(R_(A))C(═O)R_(C), —NO₂, —CN, —CF₃, —CHF₂, —CH₂F, —NH₂, —SCN,—SO₂CN, —F, Cl, —Br, —I, —PO₃H₂, —OPO₃H₂, —C_(n)H_(2n)R_(C) which isbranched or unbranched wherein n is an integer from 1 to 5;—C_(n)H_((2n−2))R_(C) in E or Z geometrical conformation which branchedor unbranched wherein n is an integer from 2 to 5; —C_(n)H_((2n−4))R_(C)which is branched or unbranched wherein n is an integer from 2 to 5;wherein R_(A), R_(A)′, R_(A)″ are each independently selected from thegroup consisting of: —H, lower alkyl group, —CN, —CF₃, —CHF₂, —CH₂F,—OH; wherein R_(B) is independently selected from the group consistingof: —H, lower alkyl group, —CN, —CF₃, —CHF₂, —CH₂F, —CH₂Cl, —CH₂Br,—CH₂I; wherein R_(C) is independently selected from the group consistingof: —H, lower alkyl group, —CN, —CF₃, —CHF₂, —CH₂F, —CH₂Cl, —CH₂Br,—CH₂I, —F, —Cl, —Br, —I, —NH₂, wherein R_(D) is independently selectedfrom the group consisting of: —H, lower alkyl group.
 3. The compositionof claim 1, wherein the compound is represented by the followingstructures:


4. The composition of claim 2, wherein the compound is represented bythe following structures:


5. The composition of claim 1, wherein the compound has an effect onmutant CFTR protein, wherein the mutant CFTR protein is a mutationΔF508-CFTR, or another mutation of class II, and wherein a mutationΔF508-CFTR, or another mutation of class II are involved in CFTR proteinmalfunction.
 6. The composition of claim 2, wherein the compound has aneffect on mutant CFTR protein, wherein the mutant CFTR protein is amutation ΔF508-CFTR, or another mutation of class II, and wherein amutation ΔF508-CFTR, or another mutation of class II are involved inCFTR protein malfunction.
 7. The composition of claim 3, wherein thecompound has an effect on mutant CFTR protein, wherein the mutant CFTRprotein is a mutation ΔF508-CFTR, or another mutation of class II, andwherein a mutation ΔF508-CFTR, or another mutation of class II areinvolved in CFTR protein malfunction.
 8. The composition of claim 4,wherein the compound has an effect on mutant CFTR protein, wherein themutant CFTR protein is a mutation ΔF508-CFTR, or another mutation ofclass II, and wherein a mutation ΔF508-CFTR, or another mutation ofclass II are involved in CFTR protein malfunction.
 9. The composition ofclaim 5, wherein the disease associated with CFTR protein malfunction iscystic fibrosis.
 10. The composition of claim 6, wherein the diseaseassociated with CFTR protein malfunction is cystic fibrosis.
 11. Thecomposition of claim 7, wherein the disease associated with CFTR proteinmalfunction is cystic fibrosis.
 12. The composition of claim 8, whereinthe disease associated with CFTR protein malfunction is cystic fibrosis.13. The compound of claim 1, wherein said compound is a modulator foruse in the treatment of cystic fibrosis wherein it has effect onCFTR-dependent ion transport across cellular membrane and/or it has theability to increase the number of mutant CFTR proteins that reach thecell surface.
 14. The compound of claim 1, wherein said compound is amodulator for use in the treatment of cystic fibrosis wherein it hasstabilizing effect on the structure of the mutant CFTR protein and/orblocks the interaction with cellular proteins responsible for thepremature degradation of mutant CFTR.
 15. The compound of claim 1,wherein said compound is a modulator for use in the treatment of cysticfibrosis wherein it has effect on mutant CFTR protein, wherein said CFTRmutation is a mutation ΔF508-CFTR, or another mutation of class II.